The invention relates to a device for electrochemically measuring biochemical reactions, a method for producing the device and a method for electrochemically measuring biochemical reactions with the aforementioned device.
Electrochemical biosensors are used for a range of applications in biosensor technology, for example to detect viruses or antibodies or in DNA analysis. A range of detection reactions require particular temperature conditions and need to be subjected to particular temperature cycles. The electrochemical biosensors therefore need to be contacted not only electrically and fluidically, but also thermally. The electrochemical sensors generally comprise metal surfaces, which are coated or uncoated and which must be stable in the solutions to be analyzed. The sensors are arranged in array form on a base plate, in order to be able to carry out parallel measurements and for analyses of a plurality of individual components simultaneously.
When a sensor array is being used, each sensor spot or electrochemical sensor must be coated with a different recognition molecule. The specific coating may be carried out by lithographic methods or by spotting. Lithographic methods are very expensive and elaborate, since lithographic masks need to be prepared for each application and the chemical reactions for the coating comprise a series of steps.
Specific coating of the sensors by spotting with solutions which contain the recognition molecules to be applied, including binding groups, is simpler and more economical. When gold electrodes are used, binding groups may comprise thiol compounds which lead to directional binding of the recognition molecules on the gold surface. The electrodes may alternatively be formed of platinum, platinum being chemically very stable in a range of solutions, although during electrochemical measurements it may lead to a potential drift in the solutions being used.
The electrodes may be arranged mechanically stably on a base plate made of glass, silicon or plastic. In this case, the metal electrodes are applied onto the base plate by thin or thick film technology and electrically contacted via webs. In the case of silicon base plates, the measurement and evaluation electronics may be contained on the base plate in the form of an integrated circuit. Silicon is very expensive, however, and the production of integrated circuits on silicon is likewise expensive and elaborate. As an alternative, the base plate may be formed of printed circuit boards (PCB). The production of these is particularly straightforward and economical. Electrical leads and contacts are formed in the manner of copper tracks on the printed circuit board and electrically insulated from the environment by a varnish. The electrodes may be formed by coating the copper e.g. with gold, when using interlayers, and uncovered by the varnish layer.
By spotting liquids with different recognition molecules onto different electrodes of an array, the electrodes can be prepared for the specific detection of different biomolecules. For each electrode, a drop of a solution of a particular recognition molecule is spotted and the recognition molecules bind to the electrode. The solvent may be removed, for example by evaporation. During the spotting, however, it is necessary to prevent the spotting solution of one electrode from spreading over the surface of the base plate so that it comes into contact with a second electrode. Spreading can lead to unintended coating of a neighboring electrode, which will then not be specifically coated with recognition molecules and will not allow specific detection. In order to prevent the spotting solution from spreading, it is possible to use hydrophobic coatings of the base plate around the electrodes. This method is elaborate, not very reliable and works only when a small amount of solution is spotted.
Alternatively, it is possible to provide indentations in which the electrodes are embedded as a base surface of the indentation. In order to provide the indentations, for example, plastic rings may be fastened around the electrodes on the base plate or structured films, having recesses at the positions of the electrodes, may be applied or adhesively bonded onto the base plate. Elevations from the surface plane of the base plate are thereby formed, which act as a coating aid. However, these coating aids may, in the event of a fluid flow over the sensors, lead to formation and fixing of air bubbles which can interfere with an electrochemical measurement and which lead to false measurement results.
Fluidic contacting of the electrochemical biosensors is carried out by fitting a flow cell which is mechanically connected to the biosensor. The flow cell has an inlet channel and an outlet channel. Liquids to be studied can thus be pumped through the flow cell, i.e. flow over the sensor array on the base plate, and in the event of specific binding of biomolecules to individual sensors of the sensor array the binding events are measured by electrochemical signals. Fluidic contacting is in this case carried out from the base plate side on which the sensor array is arranged. Thermal contacting is carried out from the side of the base plate which lies opposite the base plate side with the sensor array.
Fitting a flow cell on the base plate with the biosensors and sealing with the aid of sealing rings often leads to problems in handling and to sealing problems. Arrangements composed of biosensors on a base plate and a fitted flow cell are generally constructed in a very complicated way, with a range of individual parts which are elaborate to manufacture. In particular, microcavities produced by milling with small structure sizes, which are often used in flow cells, are elaborate to produce.